User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/06/30

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Objective

Continue trying to get BSA-intein construct cloned.

Bench work

  1. Ligation
    • Using new digested DNA
      • Previous purification could have been problematic, but hopefully the yield is higher and there is less ethanol this time.
    • Insert: gel-purified NheI/SapI digested BSA PCR from yesterday
    • Vector(His): gel-purified NheI/SapI/Phosphatased pTXB1His1 from yesterday
    • Vector: gel-purified NheI/SapI/Phosphatased pTXB1 from yesterday
    1. 8.5μL Insert + 8.5μL Vector(His) + 2μL buffer + 1μL T4 DNA Ligase
    2. 8.5μL Insert + 8.5μL Vector + 2μL buffer + 1μL T4 DNA Ligase
    3. 8.5μL Vector(His) + 8.5μL H2O + 2μL buffer + 1μL T4 DNA Ligase (negative control)
    4. 8.5μL Vector + 8.5μL H2O + 2μL buffer + 1μL T4 DNA Ligase (negative control)
    • → 16°C O/N
    • → store @ -20°C
  2. Transformation
    1. 100μL DH10B + 5μL V(His)+I from yesterday
    2. 100μL DH10B + 5μL V+I from yesterday
    3. 100μL DH10B + 5μL V(His) neg ctrl from yesterday
    4. 100μL DH10B + 5μL V neg ctrl from yesterday
      • heat shock time is 45s
      • plate 100μL each
  3. Re-plate yesterday's NovaBlue transformation
    • 450μL of each re-plated
    • → 37°C O/N
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